The long-term goal of this research is to understand the role of calcium activated neutral protease, calpain, in normal lenses and lenses undergoing cataract formation. Studies conducted in the preceding grant cycle showed: 1) Calpain and its endogenous inhibitor are present as a major protease system in human and rodent lenses. 2) Calpain is diffusely distributed across epithelial cells in central epithelium, where it may be associated with cytoskeleton. In contrast, calpain in differentiating epithelial cells and outer cortex outlined the periphery of the fibers as if it were associated with the plasma membrane. 3) Preliminary results showed that calpain inhibitor E64 was able to prevent cataract and proteolysis in cultured rat lenses. These data support the hypothesis that calpain in lens escapes inhibition of cytosolic calpastatin by binding to fiber membranes, where calpain is activated. In normal lenses, this leads to hydrolysis of cytoskeleton involved in terminal fiber cell differentiation. In cataractogenesis, breakdown of crystallins and membrane proteins by excessive action of calpain leads to insolubilization and opacity. Three experiments are proposed to further test this hypothesis: 1) Examine the nature of calpain binding to the lens membrane, and determine if binding of calpain to lens membrane allows escape from calpastatin and activation. 2) Examine the association of calpain with subcellular structures in lens using transmission electron microscopy. 3) Determine the effectiveness of new, more specific, cell penetrating inhibitors of calpain in preventing cataract and associated biochemical changes in cultured rodent lenses. The studies proposed should provide a more detailed understanding of the regulation and role of calpain activity in lens. They will also provide data for possible future testing of calpain inhibitors as drugs to ameliorate or prevent cataract in man.